Chronic ethanol exposure diminishes adenylyl cyclase activity in animals and in cells in culture. The mechanism of this change involves ethanol- induced alterations in Gsalpha, the stimulatory GTP-binding protein of adenylyl cyclase. Ethanol-induced changes in adenyly cyclase may contribute to the acquisition of tolerance and dependence to ethanol. There is evidence that susceptibility to ethanol-induced changes in adenylyl cyclase, may in part, be genetically determined. In aim I, we will characterize the lymphocyte adenylyl cyclase system in family history positive and family history negative alcoholics and in family history positive and family history negative nonalcoholics. Employing alcoholics and nonalcoholics grouped by the familial absence or presence of alcoholism allows us to clarify which defects in adenyly cyclase are due to prior ethanol abuse and which abnormalities are determined by genetic factors. In these 4 subgroups, we will study the expression of the GTP-binding proteins, Gs and Gi, by Northern and Western analysis. Both quantitative and qualitative G protein alterations will be correlated with changes in adenylyl cyclase activity. To assess functional differences in Gsalpha between subgroups, Gsalpha protein will be expressed in S49-cyc cells, which genetically lack endogenous Gsalpha and signalling function compared. Gsalpha activates the catalytic unit of adenylyl cyclase to manufacture CAMP. The cDNA for the catalytic unit of adenylyl cyclase has recently been cloned. To date, there is no published data that has examined the effects of ethanol on the expression of this mRNA. In the final part of AIM I, we will investigate the expression of this important mRNA in our four subgroups. In aim II, specific genetic studies will include analysis of genomic DNA from our 4 subgroups by restriction endogenous analysis to identify deletions, and insertions, or re-arrangements in the Gsalpha gene. to identify small mutations (eg. single base), we will use denaturing gradient gel electrophoresis to analyze specific exons and exon-intron junctions of the Gsalpha genes that have been amplified by the polymerase chain reaction. To date, we have identified a defect in exons4-5 of Gsalpha which is present in our alcoholic proband but not present in 10 controls. This region of the Gsalpha gene encodes for a domain in Gsalpha protein that interacts with the catalytic unit of adenylyl cyclase. Sequencing is underway to determine if this abnormality is a neutral polymorphism or a functional mutation. Lastly, in AIM III, we shall determine if an adenyly cyclase-regulated hormonal axis can be a marker for a predisposition for alcoholism. To this end, we will compare the dynamics of the Pituitary-Adrenal axis in family history positive and family history negative non-alcoholics.